The present invention relates to a polypeptide antigen which derives from the gp41 protein, and also to its use for immunization against HIV-related infections; these studies were cofinanced by the ANRS [French National Association for AIDS Research].
The development of a method of immunization against HIV is, to lay, one of the priorities of scientific research.
The major obstacles represented by the great genetic variability of the virus and the low exposure to the immune system of neutralizing viral epitopes considerably hinder the development of neutralizing immunity.
The HIV envelope glycoprotein, which is required to confer on the virus its infectious nature, represents the target for neutralizing antibodies. These characteristics have made this glycoprotein a subject of intense investigation.
The envelope glycoprotein (env) of the human immunodeficiency virus-1 (HIV-1) is synthesized from the precursor gp160, which gives, under the action of a protease, the gp120 and gp41 subunits.
The attachment of gp120/gp41 to the cellular receptors induces a change in conformation of gp41, from a latent (nonfusogenic) state to an active-fusion (fusogenic) state. Between these two states, there exists a transient xe2x80x9cintermediatexe2x80x9d state, during which gp41 is like a membrane-bound protein which is in both the viral and cell membranes (Weissenhorn et al. Nature (1997), 387 (6631), 426-30).
The use of gp41 in its fusogenic conformation, for immunization purposes, is described in WO 00/40616. According to that application, the N-helices can be used alone or in combination with the C-helices so as to reproduce, in the latter case, the fusogenic conformation of gp41.
Binding experiments have made it possible to establish, firstly, that the nonfusogenic latent state is characterized by the inaccessibility of large portions of the ectodomain of gp41. gp120 in fact interacts so as to mask the epitopes. It has, moreover, been shown that inhibition of the change in structure of the intermediate state to the fusogenic state with peptides used as competitors may affect viral infection (Weissenhorn W. et al., Molecular Membrane Biology, 1999, 16, 3-9).
The applicant proposes a novel polypeptide antigen which can be used for therapeutic and prophylactic immunization against HIV-related infections. The applicant has, in fact, demonstrated, for the first time, that a polypeptide which mimics the intermediate state of gp41 is capable of inducing antibodies which neutralize primary isolates of HIV.
The present invention therefore relates to a polypeptide comprising a sequence of formula I:
[Nxe2x88x92S1]nxe2x88x92Cxe2x88x92[S2xe2x88x92N]m
in which:
N represents the sequence of amino acids 30 to 77 of gp41,
C represents the sequence of amino acids 117 to 154 of gp41,
S1 and S2 are, independently of one another, either absent or represent an amino acid sequence such that the sequence of formula I adopts an alpha-helical conformation as determined by the SOPMA program under the following conditions:
number of conformational states=4
similarity limit=8, and
window width=70
n=0 or 1; m=0 or 1 and m+n=1 or 2.
Preferably, the polypeptide as defined above comprises a sequence of formula I in which m+n=1.
Preferably, S1 is absent or represents the amino acid sequence D, DQ, DQQ, DQQL or DNNMT, and S2 is absent or represents the amino acid sequence W, WA, WAS, WASL or WASLW.
The S1 and S2 amino acid sequences are defined using the one letter code in which D represents aspartic acid, Q represents glutamine, etc.
According to a particular embodiment, the polypeptide comprises a sequence of formula I as defined above, in which N represents SEQ ID No. 26 and C represents SEQ ID No. 27.
According to a preferred embodiment, the polypeptide is selected in the group consisting of SEQ ID No. 28 and SEQ ID No. 31.
According to another embodiment, the polypeptide according to the invention comprises an additional sequence of formula (G)a-Sxe2x80x94(H)b in which G represents a glycine residue, H represents a histidine residue S is a serine, a is greater than or equal to 4 and b is greater than or equal to 6. Said sequence is linked via an amide bond to the NH2-terminal end or COOH-terminal end of the polypeptide according to the invention.
According to another aspect, the present invention relates to a conjugate comprising a polypeptide according to the invention conjugated to a carrier protein or peptide.
According to another aspect, the present invention relates to a DNA sequence encoding a polypeptide according to the invention or a conjugate according to the invention.
The present invention also relates to an expression vector comprising said DNA sequence, and also to a host cell containing said vector.
A subject of the present invention is also a pharmaceutical composition comprising at least one polypeptide as defined above, at least one conjugate as defined above or at least one expression vector as defined above, a pharmaceutically acceptable excipient and, optionally, an adjuvant.
According to a particular embodiment, the present invention relates to a pharmaceutical composition as defined above which can be administered orally.
A subject of the present invention is therefore also the polypeptide as defined above, for its use as a medicinal product, and in particular for its use in immunizing the human body against HIV-related infections.
Another subject of the present invention relates to the method for preparing a polypeptide as described above, comprising the expression of said polypeptide using a host cell as defined above.
The invention is described in greater detail in the description which follows.
The phenomenon of conformational change of gp41 which precedes the cell and viral membrane fusion is illustrated in FIG. 1. Attachment of gp120 to the receptor and the coreceptor of the virus causes a first modification which leads to unfolding of the fusion peptide and anchoring thereof in the cell membrane. At this time, an intermediate state forms, in which there is no interaction between the N-helices and the C-helices. The second event illustrated in FIG. 1 is the folding of gp41, which corresponds to the molecule adopting a thermodynamically more stable conformation. The energy released during this folding allows the lipids of the cell and viral membranes to come close to one another and fuse.
The Applicant has demonstrated, surprisingly, that the polypeptide according to the invention induces specific IgG antibodies which neutralize HIV primary isolates. The induction of antibodies which neutralize primary isolates can be determined using the neutralization test as described in the article by C. Moog et al. (AIDS Research and human retroviruses, Vol. 13(1), 13-27, 1997), to which reference may be made for a complete description of the latter. In the context of the present invention, it is estimated that neutralizing antibodies have been induced by the antigen tested according to the technique of C. Moog when the serum diluted at least to xc2xc, in the presence of HIV, leads to a 10-fold decrease in the viral titer in comparison to HIV alone, the viral titer being evaluated by the amount of p24 produced in the culture supernatant.
The induction of antibodies which neutralize primary isolates may also be determined using the neutralization test of D. Montefiori as described in J. Infect. Dis. 1996, 173:60-67. In this test, the neutralizing titer is expressed by the percentage decrease in p24 antigen produced in the culture supernatants when the virus is incubated in the presence of serum diluted to xc2xc, by comparison with the virus in the absence of serum. In the context of the present invention, it is considered that neutralizing antibodies have been induced when the decrease in the level of p24 produced reaches at least 80% with a serum diluted to xc2xc.
In the context of the present invention, it is considered that the antibodies induced by the polypeptide according to the invention are neutralizing antibodies if neutralizing activity is detected for a given isolate in at least one of the two tests above.
The induction of antibodies which neutralize the HIV-1 MN laboratory strain can be estimated using the MT-2 cell line according to the method of D. Montefiori, described in: DC Montefiori et al., J. Clin. Microbiol. 1988, 26: 231-5). In this method, the neutralizing titer is expressed as the inverse of the dilution of the serum (in arithmetic value) which protects at least 50% of cells against the cytopathogenic effect of the HIV virus.
This property makes the polypeptide according to the invention an immunization candidate of interest for humans.
The polypeptide according to the present invention has the particularity of adopting, under physiological conditions, a conformation which may be termed xe2x80x9copenxe2x80x9d, as opposed to the xe2x80x9cclosedxe2x80x9d conformation of gp41 in fusogenic form in which the N and C domains are paired with one another according to an anti-parallel orientation (cf. FIG. 1).
Specifically, the open conformation of the polypeptide according to the invention is characterized in that the C sequence is not paired with the N sequence according to the anti-parallel orientation as present in the fusogenic form. The polypeptide according to the present invention is preferably in a trimeric form in which the N sequences and the C sequences are, respectively, paired with one another according to a parallel orientation.
The applicant has also demonstrated, surprisingly, that the polypeptide according to the invention conserves its xe2x80x9copenxe2x80x9d conformation in highly acid medium. This property makes the polypeptide according to the invention an immunization antigen which can be administered orally. The applicant has, in fact, shown that the ectodomain of the gp41 protein is extremely thermostable at pH 2.5.
Measurement of the Tm (temperature at which 50% of the proteins present are denatured) of gp41 in 50 mM sodium formate by DSC (differential scanning calorimetry) gives a value of 110xc2x0 C., the beginning of the denaturation phenomenon appearing at approximately 100xc2x0 C. at pH=2.5. The thermostability of the gp41 protein at neutral pH has been evaluated by Weissenhorn et al. (EMBO, 1996, 7, 1507-1514). The Tm measured at neutral pH by these authors is 78xc2x0 C., which means that, surprisingly, this protein is more stable at acid pH than at neutral pH. These results have been confirmed by circular dichroism analysis aimed at calculating the percentage of alpha-helix in the protein.
The applicant has also shown that the polypeptide according to the invention has the same particularity. This specific property makes the polypeptide according to the invention an immunization antigen of choice for oral administration.
This open conformation is obtained by the direct attachment of the N and C sequences as defined above. In such a case, S1 and S2 are absent and the last amino acid of the N sequence is directly linked, via an amide bond, to the first amino acid of the C sequence, or inversely (example: AA33-77-AA117-154). This open conformation is also obtained when the N and C sequences as defined above are connected to one another via an S1 or S2 sequence such that the resulting sequence of formula I adopts an alpha-helical conformation as determined by the SOPMA program under the following conditions: number of conformational states=4; similarity limit=8 and window width=70.
The open conformation according to the invention is, in fact, characterized by the absence of a flexible region between the N and C sequences. The SOPMA program available on the site: http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html (Geourjon C and Delxc3xa9age G. SOPMA, Casios (1995), 11, 681-684) makes it possible to easily determine whether the S1 or S2 sequence adopts, in the context of the polypeptide of formula I, an alpha-helical conformation which is suitable in the context of the present invention.
According to a preferred embodiment, m+n=1 and, if S1 is absent, S2 represents W, WA, WAS, WASL or WASLW and, if S2 is absent, S1 represents D, DQ, DQQ, DQQL or DNNMT.
In addition, according to a particularly preferred embodiment, in the polypeptide as defined above, the sequences AA30-AA77 and AA117-AA154 correspond, respectively, to the sequences SEQ ID No. 26 and SEQ ID No. 27.
According to a preferred embodiment, the polypeptide is selected in the group consisting of SEQ ID No. 28 and SEQ ID No. 31.
The sequences AA30-AA77 and AA117-AA154 derived from gp41 are numbered by taking, as a reference, the numbering of the sequence of the gp41 protein as given in the sequence listing under the title SEQ ID No. 25. The sequence SEQ ID No. 25 corresponds to a truncated gp41 LAI protein in which the first amino acid A bears the number 1.
The sequences AA30-AA77 and AA 117-AA154 according to the present invention may be derived from any gp41 protein of HIV, including the HIV1 and HIV2 strains, including the laboratory strains and primary isolates. Preferably, the N and C sequences according to the present invention are derived from an HIV1 strain, and in particular from an HIV1 LAI strain.
The nucleotide and peptide sequences of a large number of gp41 proteins are known and available, for example, on the Internet on the site http://hiv-web.lanl.gov/, and also in the corresponding Los Alamos compendia. It is clear that any sequence into which one or more conservative mutations which do not substantially modify either the immunogenicity or the open conformation have been introduced is also included in the context of the present invention.
The open conformation according to the present invention is characterized by a certain number of parameters which can be measured by the DSC technique and the circular dichroism technique. These techniques are described in detail in the following articles by A. Cooper et al., Phil Trans. R. Soc. Lon. A (1993) 345, 23-25, and by V. V. Plotnikov et al. Analytical Biochemistry 250, 237-244, (1997) and S. M. Kelly and N. C. Price (1997) Biochim. Biophys. Acta. 1338, 161-185.
DSC measures the thermodynamic parameters of protein denaturation. Protein denaturation is an endothermic event which can be evaluated by measuring the absorption of heat by the protein as a function of temperature. The two main parameters obtained are:
the Tm or half-denaturation point (i.e. temperature at which 50% of the protein is present in native form and 50% in denatured form) and
the xcex94H or variation in enthalpy during denaturation (i.e. the heat required to denature the protein).
These two parameters are precise markers of the conformation of a protein. A DSC analysis is detailed in example 5 with reference to FIG. 2.
As regards the circular dichroism technique, it makes it possible to evaluate the secondary structure of the protein. The signal for an alpha-helix is characterized by minima at 208 nm and 222 nm. The value of the signal at 222 nm is used to determine the percentage of alpha-helix in a protein. The signal for a loop is characterized by a maximum at approximately 205 nm. This technique makes it possible to demonstrate that the polypeptide according to the invention does not contain the loop normally present in the fusogenic form and that it consists almost exclusively, or even exclusively, of an alpha-helix.
A circular dichroism analysis is detailed in example 6 with reference to FIG. 3.
Although the polypeptide according to the invention has an open conformation which is stable, this conformation may be reinforced by adding cysteine residues to the ends of the polypeptide. To this end, two additional cysteine residues may be added at the N-terminal or at the C-terminal, preferably at the N-terminal, of the polypeptide according to the invention so as to covalently fix the trimer in an open conformation.
The polypeptide according to the invention may be obtained using any conventional technique of chemical synthesis or of genetic engineering.
When the polypeptide is produced by chemical synthesis, the polypeptide according to the invention may be synthesized in the form of a single sequence, or in the form of several sequences which are then linked to one another. The chemical synthesis may be carried out in solid phase or in solution, these two synthesis techniques being well known to those skilled in the art. These techniques are described in particular by Atherton and Shepard in xe2x80x9csolid phase peptide synthesis (IRL press Oxford, 1989) and by Houbenweyl in xe2x80x9cmethod der organischen chemiexe2x80x9d edited by E. Wunsch vol, 15-I and II thieme, Stuttgart, 1974, and also in the following articles, which are entirely incorporated herein by way of reference: Dawson P E et al. (Synthesis of proteins by native chemical ligation Science 1994; 266(5186):776-9); Kochendoerfer G G et al. (Chemical protein synthesis Curr Opin Chem Biol 1999; 3(6):665-71); and Dawson P E et al. Synthesis of native proteins by chemical ligation, Annu rev Biochem 2000; 69:923-60.
The polypeptide according to the invention may also be produced using genetic engineering techniques which are well known to those skilled in the art. When the polypeptide according to the invention is produced by genetic engineering, it comprises an additional NH2-terminal methionine residue corresponding to the translation of the first initiation codon and it may further comprises additional N-and/or C-terminal amino-acid(s). These techniques are described in detail in Molecular Cloning: a molecular manual by Maniatis et al., Cold Spring Harbor, 1989). Conventionally, the DNA sequence encoding the polypeptide according to the invention may be produced by the PCR technique, in which the N and C sequences are, firstly, amplified independently of one another and are then, secondly, paired and again amplified. The DNA sequence thus obtained is then inserted into an expression vector. The expression vector containing the sequence of interest is then used to transform a host cell which allows the expression of the sequence of interest. The polypeptide produced is then isolated from the culture medium using conventional techniques well known to those skilled in the art, such as ethanol precipitation or ammonium sulfate precipitation, acid extraction, anion/cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography or lectin chromatography. Preferably, high performance liquid chromatography (HPLC) is used in the purification.
Depending on the expression system used (secreted or nonsecreted protein) and depending on the purification method, the purified polypeptide may be in various forms. It may be in a denatured or nondenatured, monomeric or multimeric form. When it is in a denatured form, it is possible to turn it to its open conformation according to the invention using the method described in the examples given hereinafter. To obtain multimeric forms, and in particular trimers, the purified polypeptide molecules must be placed in a medium which allows the molecules to be completely soluble and to have essentially no interaction with one another and preferably no secondary structure. For this, it is possible to use detergents such as sodium dodecyl sulfate, N-lauryl sarcosine, guanidinium chloride, urea, sodium thiocyanate or chaotropic agents. The desired conditions may be promoted by using organic solvents or using acids. Once this first condition is satisfied, the sample is placed in a dialysis cassette in order to remove part of the detergents or of the chaotropic agents used, so as to promote the interactions between the polypeptide monomers while conserving sufficient solubility for the molecules. In a second step, once the formation of trimers has been promoted, the sample is completely dialyzed in a physiological medium which keeps the polypeptide in solution or in suspension. Trimers of the polypeptide according to the invention are thus obtained. Such a technique is described in detail in WO 00/08167.
To carry out the synthesis of the polypeptide, any expression vector conventionally used for expressing a recombinant protein may be used in the context of the present invention. This term therefore encompasses both xe2x80x9clivexe2x80x9d expression vectors, such as viruses and bacteria, and expression vectors of the plasmid type.
Vectors in which the DNA sequence of the polypeptide according to the invention is under the control of a strong promoter, which may be inducible or noninducible, are preferably used. By way of example of a promoter which may be used, mention may be made of the T7 RNA polymerase promoter.
The expression vectors preferably include at least one selection marker. Such markers include, for example, the dihydrofolate reductase gene or the neomycin resistance gene, for culturing eukaryotic cells, and the kanamycin resistance, tetracycline resistance or ampicillin resistance genes, for culturing in E. coli and other bacteria.
By way of an expression vector which may be used in the context of the present invention, mention may be made of the pET28 (Novagen) or pBAD (Invitrogen) plasmids for example, viral vectors such as: baculoviruses, poxviruses, in particular the poxviruses described in patents U.S. Pat. Nos. 5,942,235, 5,756,103 and 5,990,091, which are entirely incorporated herein by way of reference, and recombinant vaccinia viruses, in particular the recombinant viruses described in patents EP 83286, U.S. Pat. Nos. 5,494,807 and 5,762,938, in which the DNA sequence encoding a polypeptide according to the invention is cloned.
To promote the expression and the purification of the polypeptide, the latter may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminal of the polypeptide to improve stability and persistence in the host cell.
For expression of the polypeptide, any host cell conventionally used in combination with the expression vectors described above may be used.
By way of nonlimiting example, mention may be made of the E. coli cells BL21 (xcexDE3), HB101, Top 10, CAG 1139, Bacillus, and eukaryotic cells such as CHO or Vero.
In the context of the present invention, use will preferably be made of the following expression vector/cell system: pET(Cer)/BL21LambdaDE3 or BL21-lambdaDE3(RIL).
Depending on the host cell used for expressing the polypeptide, the polypeptides of the present invention may be glycosylated or nonglycosylated. In addition, the polypeptides according to the invention may also include an additional N-terminal methionine residue as well as some additional N- or C-terminal amino acid residues resulting from the recombination process.
Once purified, the polypeptide according to the invention may advantageously be mixed with 2,2,2-trifluoroethanol (TFE). To do this, the polypeptide is preferably placed in an acid buffer, such as sodium formate at pH=2.5. The mixture thus formed contains from 10 to 50% by volume of TFE, preferably from 15 to 30% by volume of TFE. The effect of the TFE is to increase the degree of helicity of the polypeptide so as to take it to a value close or equal to 100%.
Besides the TFE, other organic solvents or detergents may also be used. By way of example, mention may be made of: isopropanol or lysophospholipid. The suitable amounts of these compounds may be easily determined by those skilled in the art. Since these compounds are in general toxic and, consequently, cannot be administered to humans, it is necessary to eliminate them before administration. The applicant has demonstrated that these solvents or detergents may be easily eliminated by adding a support which adsorbs the polypeptide. The support used in the context of the present invention corresponds to a pharmaceutically acceptable support which may be administered to humans. By way of example of a support which may be used in the context of the present invention, mention may be made of the aluminum hydroxide, phosphate or hydroxyphosphate gels which are conventionally used as adjuvants in vaccines. The support is used in large excess compared to the polypeptide so as to obtain total adsorption of the latter.
Any other pharmaceutically acceptable support capable of adsorbing the polypeptide according to the invention may be used in the context of the present invention.
Conventionally, the required amount of pharmaceutically acceptable support is added to the polypeptide/TFE mixture and the entire mixture is then incubated at room temperature for the amount of time necessary to allow total absorption of the polypeptide onto the support. The incubation period may vary from 15 min to 3 h. The mixture is then centrifuged once or twice at approximately 10 000 g and the pellet is taken up in a solution which can be administered to humans, such as for example in the case of an injectable composition of the PBS buffer or a physiological saline solution.
A subject of the present invention is also the conjugates comprising a polypeptide according to the invention and a carrier protein or a carrier peptide.
The carrier protein (or peptide) strengthens the immunogenicity of the polypeptide according to the invention, in particular by increasing the production of specific antibodies. Said carrier protein (or peptide) preferably comprises one or more T helper epitope(s). The term xe2x80x9cT helper epitopexe2x80x9d is intended to mean a chain of amino acids which, in the context of one or more class II MHC molecules, activates T helper lymphocytes. According to an advantageous embodiment, the carrier protein (or peptide) used improves the water-solubility of the polypeptide according to the invention.
As carrier protein, use may be made, for example, of phage surface proteins, such as the pIII or pVIII protein of the M13 phage, bacterial surface proteins, such as the LamB, OmpC, ompA, ompF and PhoE proteins of E. coli, the CotC or CotD protein of B. subtilis, bacterial porins, such as Neisseria gonorrheae porin P1, H. influenzae B porin P1 or P2, N. meningitidis B class I porin or K. pneumoniae porin P40, lipoproteins, such as B. bugdorfi OspA, S. pneumoniae PspA, N. meningitidis B TBP2, E. coli TraT and also S. pneumoniae adhesin A, and the heat shock proteins, such as Hsp65 or Hsp71 of M. tuberculosis or bovis, or Hin 47 of H. influenzae type B. Detoxified bacterial toxins, such as the tetanus or diphtheria toxoid, the cholera toxin B subunit, the B subunit of P. aeruginosa endotoxin A or S. aureus exotoxin A.
In the context of the present invention, as a carrier peptide, use may be made, for example, of the p24E, p24N, p24H and p24M peptides described in WO 94/29339 and also the PADRE peptides as described by Del guercio et al. (Vaccine (1997); vol 15/4, p 441-448).
The carrier protein (or peptide) is linked to the N- or C-terminal end of the polypeptide according to the invention using any conjugation method well known to those skilled in the art. In addition, the sequence encoding the carrier protein (or peptide) may advantageously be fused to the sequence encoding the polypeptide according to the invention, and the resulting sequence may be expressed in the form of a fusion protein using any conventional method. All the genetic engineering techniques which are useful for doing this are described in Maniatis et al. Said conjugates may be isolated using any conventional purification method well known to those skilled in the art.
A subject of the present invention is also the DNA sequences encoding the polypeptides and the conjugates according to the invention, and also the expression vectors comprising said sequences and the host cells transformed with said sequences. The DNA sequences encoding the polypeptides according to the invention may be easily produced by PCR using, as a matrix, the nucleotide sequence of a gp41 protein.
Rather than extracting and purifying the polypeptide or the conjugate expressed by the expression vector, it is often easier and sometimes more advantageous to use the expression vector itself in the vaccine according to the invention. A subject of the present invention is therefore any expression vector as defined above. In such a situation, the expression vector lacks a marker and preferably corresponds to a viral vector, in particular a poxvirus such as ALVAC or NYVAC.
Any host cell as defined above transformed with an expression vector is also included in the context of the present invention.
A subject of the present invention is also the antibodies directed against the polypeptides and conjugates as described above. The preparation of such antibodies is carried out using conventional techniques for producing polyclonal and monoclonal antibodies, well known to those skilled in the art.
These antibodies are particularly suitable for use in a passive immunization scheme.
A subject of the present invention is also pharmaceutical compositions which are of use for inducing HIV neutralizing antibodies which are useful for the purposes of therapeutic and prophylactic immunization against HIV-related infections. The compositions according to the present invention comprise at least one polypeptide, at least one conjugate or at least one expression vector as defined above, a pharmaceutically acceptable diluent or excipient and, optionally, an adjuvant.
According to a preferred embodiment, the composition according to the invention also comprises a pharmaceutically acceptable support. Any pharmaceutically acceptable support capable of adsorbing the polypeptide according to the invention may be used. A description of such supports has been provided above. When the pharmaceutically acceptable support is an aluminum salt, the latter performs both the function of support and that of adjuvant.
The amount of polypeptide, of conjugate or of vector in the composition according to the present invention depends on many parameters, as will be understood by those skilled in the art, such as the nature of the carrier protein, the vector used or the route of administration. A suitable amount is an amount such that a humoral immune response capable of neutralizing primary isolates of HIV is induced after administration of this amount. The amount of polypeptide to be administered is of the order of 10 xcexcg to 1 mg, the amount selected varying depending on the route of administration. The amount of conjugate to be administered will be deduced from the amounts indicated above, taking into account the MW of the carrier protein. The amount of expression vector to be administered is of the order of 10 to 5000 micrograms in the case of a nonviral vector, and of the order of 10E4 to 10E8 TCID50 in the case of a viral vector.
The pharmaceutical compositions according to the present invention may also contain an adjuvant. Any pharmaceutically acceptable adjuvant or mixture of adjuvants conventionally used in the field of vaccines may be used for this purpose. By way of example, mention may be made of aluminum salts, such as aluminum hydroxide or aluminum phosphate. Conventional auxiliary agents, such as wetting agents, fillers, emulsifiers, buffers, etc., may also be added to the composition according to the invention.
The compositions according to the present invention may be prepared using any conventional method known to those skilled in the art. Conventionally, the antigens according to the invention are mixed with a pharmaceutically acceptable diluent or excipient, such as water or phosphate buffered saline solution. The excipient or diluent will be selected as a function of the pharmaceutical form chosen, of the method and route of administration, and also of pharmaceutical practice. Suitable excipients or diluents, and also the requirements in terms of pharmaceutical formulation, are described in detail in Remington""s Pharmaceutical Sciences, which represents a reference work in this field.
The compositions mentioned above may be administered via any conventional route usually used in the field of vaccines, such as the parenteral (intravenous, intramuscular, subcutaneous, etc.) route. In the context of the present invention, intramuscular administration will preferably be used for the injectable compositions. Such an administration may advantageously take place in the thigh or arm muscles. The compositions according to the present invention may also advantageously be administered orally. Administration via the nasal, vaginal or rectal mucosa may also be recommended in the context of the present invention. The administration may also be carried out by giving a single dose or repeated doses, for example on D0 and at 1 month, 3 months, 6 months and 12 months. Injections at J0 and at 1 month and 3 months, with a booster, the periodicity of which may easily be determined by the treating physician, will preferably be used.
The pharmaceutical composition according to the present invention may advantageously be administered according to a dosage scheme comprising the co-administration of an expression vector according to the invention and of a polypeptide according to the invention, or according to a xe2x80x9cprime-boostxe2x80x9d scheme in which the vector according to the invention is administered first and the polypeptide is administered as a booster injection. In these two dosage schemes, the expression vector according to the invention may be replaced with any expression vector comprising furthermore one or more HIV antigens or epitopes other than the polypeptide according to the invention, and in particular with a poxvirus, preferably ALVAC or NYVAC. By way of examples of ALVAC and NYVAC vectors which can be used for this purpose, mention may be made of the vectors described in patents U.S. Pat. Nos. 5,942,235, 5,756,103 and 5,990,091; EP 83286, U.S. Pat. Nos. 5,494,807 and 5,762,938. In the context of the compositions which can be administered orally, bacterial vectors such as lactobacillus or salmonella may also advantageously be used. The use of these bacterial vectors for immunization purposes is described in detail in International Journal of Food Microbiology 41 (1998) 155-167 by P. H. Pouwels et al. and Cell vol 91, 765-775, Dec, 1997 by A. Darji et al., to which reference may be made for greater detail.
The present invention is also intended to cover a polypeptide, a conjugate or a vector as defined above, and the pharmaceutical composition containing these compounds, for their use as a medicinal product, in particular for inducing HIV neutralizing antibodies useful for prophylactic and therapeutic immunization of the human body against HIV-related infections.
According to a preferred aspect, a subject of the present invention is the use of a polypeptide according to the invention for immunizing the human body. The present invention therefore preferably relates to a method for administering said polypeptide so as to induce a specific humoral response.
The present invention thus provides a method for inducing HIV neutralizing antibodies comprising administration of a quantity of a pharmaceutical composition as defined above which is sufficient to induce the said humoral response.
According to a preferred embodiment, the method comprises the administration of a composition comprising SEQ ID No. 28 or SEQ ID No. 31.
The expression xe2x80x9ca specific humoral responsexe2x80x9d is intended to mean a response comprising the production of antibodies directed specifically against the polypeptide according to the invention. The production of specific antibodies may be easily determined using conventional techniques well known to those skilled in the art, such as ELISA, RIA or western blot.
The applicant has demonstrated, surprisingly, that the polypeptide according to the invention is capable, after administration, of inducing antibodies capable of neutralizing primary isolates of HIV. These antigens therefore represent candidates of value for developing a vaccine which can be used for the protection and/or treatment of a large number, or even all, of the individuals at risk from or infected with HIV.
Without wishing to be bound by any theory, the applicant thinks that the xe2x80x9copenxe2x80x9d conformation of the polypeptide according to the invention makes accessible gp41 domains which are accessible, during the phenomenon of viral and cell membrane fusion, only in the intermediate conformation which is transiently adopted. These domains made accessible would constitute the target of the antibodies induced by the polypeptide according to the invention, these antibodies thus blocking the membrane fusion phenomenon at a prefusogenic stage. The open conformation adopted by the polypeptide according to the invention is therefore thought to mimic the conformation adopted by the intermediate state.
A subject of the invention is also a diagnostic method comprising bringing a polypeptide according to the invention into contact with a biological sample and detecting the antibody/polypeptide complexes which are formed. HIV+ individuals have, in fact, anti-gp41 serum antibodies. An immunoassay (such as an ELISA assay in which the polypeptide according to the invention is attached to the assay plate and then brought into contact with the serum to be tested, and the antibody/polypeptide complexes are then detected) would therefore make it possible to diagnose infected individuals.